LPS-induced MCP-1 expression in human microvascular endothelial cells is mediated by the tyrosine kinase, Pyk2 via the p38 MAPK/NF-kappaB-dependent pathway.
Bacterial endotoxin (lipopolysaccharide or LPS) has potent pro-inflammatory properties and acts on many cell types including endothelial cells. Secretion of the CC chemokine, MCP-1 (CCL2) by LPS-activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in LPS-induced MCP-1 production in endothelial cells is not well understood. Using human microvascular endothelial cells (HMVEC), we analyzed the involvement of the non-receptor tyrosine kinase, Pyk2, in LPS-mediated MCP-1 production. There was a marked activation of the non-receptor tyrosine kinase, Pyk2, in response to LPS. Inhibition of Pyk2 activity using a pharmacological inhibitor, Tyrphostin A9 significantly attenuated LPS-induced Pyk2 tyrosine phosphorylation, p38 MAP kinase (MAPK) activation, NF-kappaB activation, and MCP-1 expression. Furthermore, specific inactivation of Pyk2 activity by transducing microvascular endothelial cells with catalytically inactive Pyk2 mutant (AAV-Pyk2MT) or Pyk2-specific siRNA significantly blocked LPS-induced MCP-1 production. The supernatants of these LPS-stimulated cells with attenuated Pyk2 activity demonstrated decreased trans-endothelial monocyte migration in comparison to LPS-treated controls, thus confirming the inhibition of functional MCP-1 production. In summary, our data suggest a critical role for the Pyk2 mediated pathway involving p38 MAP kinase and NF-kappaB in LPS-induced MCP-1 production in human microvascular endothelial cells.
Anand
Appakkudal R
AR
Department of Pathology, Ohio State University Medical Center, Columbus, OH 43210, USA.
Bradley
Ritu
R
Ganju
Ramesh K
RK
D002465:Cell Movement / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D042783:Endothelial Cells / Q000276:immunology* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051418:Focal Adhesion Kinase 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects* / Q000235:genetics / Q000276:immunology; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D020935:MAP Kinase Signaling System / Q000187:drug effects* / Q000235:genetics / Q000276:immunology; D009000:Monocytes / Q000276:immunology / Q000378:metabolism; D009154:Mutation; D016328:NF-kappa B / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D018805:Sepsis / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
C111835:CCL2 protein, human; D018932:Chemokine CCL2; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D020032:Tyrphostins; C406122:tyrphostin A9; D051418:Focal Adhesion Kinase 2; D048051:p38 Mitogen-Activated Protein Kinases